Project Summary: Lipid Biomarker Efflux from the Brain following TBI, LaPlaca, M.C., Fernndez, F.M. Traumatic brain injury (TBI) is a major health problem in the US and worldwide, affecting at least 2.5 million Americans every year. TBI is extremely variable from person to person, making the standardization of injury classification and diagnosis challenging. Fluid biomarkers can potentially provide personalized information, yet none are currently approved for TBI diagnosis or prognosis. Most of the TBI biomarker candidates are proteins that are likely released from dying neurons and glial cells, yet the brain has a very high lipid content compared to other organs, and is vulnerable to oxidative stress, leaving brain lipids extremely vulnerable to free radical attack. Furthermore, lipids may leak out of the brain more readily than proteins, rendering them ideal candidates for peripheral diagnostics. The route for biomarker clearance from the brain to the blood has been assumed to be primarily via the blood brain barrier (BBB), however a glymphatic route for molecule clearance is now recognized, changing the focus from BBB-only transport to one of potentially multiple efflux routes. Given the promise of TBI-specific lipid biomarkers and the unknown dynamics of lipid biomarker release, there is a need to better understand lipid efflux routes following TBI. The objective of the proposed work is to identify novel lipid biomarkers for TBI diagnosis and the routes by which they exit the brain as a function of time and injury severity. The overall hypothesis is that TBI-specific lipids pass through BBB and glymphatic routes to the blood, and that these efflux kinetics will vary based on injury severity and lipid properties. Three mutually- informing aims will be pursued: Aim1) Identify a serum lipid biomarker panel that is reflective of TBI severity and pathology. It is hypothesized that the number and population of altered lipids in the brain and blood will indicate injury severity; Aim 2) Determine brain efflux routes and temporal efflux dynamics of lipids after TBI. It is hypothesized that more than one route of brain clearance of TBI-generated lipid biomarkers exists and the efflux dynamics depend on lipid size and time post-injury; and Aim 3) Develop a model to predict TBI severity based on serum lipid biomarker levels. It is hypothesized that a compartmental model using parameters from Aims 1 and 2 will allow prediction of TBI severity from lipid biomarker profiles in serum. Discovery metabolomics techniques identifying lipids in serum that successfully discriminates between injured and uninjured rats will be expanded to include surveillance in cerebrospinal fluid and lymph in the acute and subacute time post-TBI for a range of injury severities in both male and female rats. Through this research, we expect to identify novel lipid biomarker panels and determine the major route(s) for their release from the brain. This is significant and novel because, while biomarkers provide a unique window into secondary injury events, changes in efflux patterns directly impact clinical interpretation and implementation.